Recently, live candidate dengue vaccines have been prepared by repeated passage of virus in cultured cells of an unnatural host. Scientists in Thailand claimed that a DEN2 vaccine derived by serial passages in primary dog kidney cells was satisfactorily attenuated for susceptible humans. Live dengue virus vaccines of other serotypes were similarly prepared and claimed to be attenuated. However, these observations have not been confirmed and experience from other laboratories indicated that this approach did not yield satisfactorily attenuated vaccine strains. Thus, a safe and effective dengue vaccine is still not available, despite five decades of intensive research. The cumulative experience gained during our molecular studies has shown that it is possible to produce a panel of dengue virus mutants that are restricted in viral replication. Also, experience with several well-studied live vaccines directed against other viruses indicates that reduced viral replication is usually associated with attenuation for humans. For this purpose, a series of DEN4 mutants containing a deletion in the 5' or 3' noncoding (NC) region that results in reduced replicative capacity in simian cell culture have been constructed and analyzed. Deletion mutants should have the advantage of being less subject to reversion of phenotype than amino acid substitution mutants. One or more mutants developed during this project might prove to be useful for immunization of humans against disease caused by dengue virus. Recently, we described construction of intertypic chimeric cDNA by replacing the C-PreM-E or only the PreM-E structural protein genes of full-length DEN4 cDNA with the corresponding genes of DEN1 or DEN2. Viable intertypic dengue chimeras that express the antigenicity of DEN1 or DEN2 were recovered from cells transfected with the cDNA-derived RNA transcripts. We showed that chimeric dengue viruses infected rhesus monkeys and were highly immunogenic when given individually or simultaneously as a bivalent vaccine. These chimeras were effective in inducing protective immunity against challenge with dengue virus of the same serotype. More recently, a viable DEN3/DEN4 chimera was also constructed using DEN4 cDNA. These encouraging observations suggest that a live multivalent dengue vaccine containing DEN4 and DEN1/DEN4, DEN2/DEN4 and DEN3/DEN4 chimeras might be feasible. The use of all four dengue virus serotypes on the same DEN4 background represents a novel dengue vaccine strategy.